Field of the Disclosure
The present invention relates to bioengineering, more particularly, to compositions for gene therapy with Vascular Endothelial Growth Factor (VEGF) having a high stability and prolonged and reliable VEGF transgene expression and a method for extending the lifetime of a transgene mRNA in a mammalian cell transfected with the composition.
Description of the Related Art
VEGF and its Biological Role
VEGF is a family of biologically active proteins first isolated by J. Folkman et al. in 1971 [8] that are produced by the cells of most or all bodily tissues, including epithelial tissue. The VEFG family is considered to represent major autocrine and paracrine factors for regulation of vasculogenesis, angiogenesis (VEGF-A, VEGF-B; PIGF) and lymphogenesis (VEGF-C, VEGF-D).
The formation of vasculature during human postnatal development of a human is mostly influenced by all VEGF isoforms including VEGF-A isoforms 121, 145, 148, 165, 183, 189 and 206 [9].
Three types of VEGF receptors have been identified. Types 1 and 2 are involved in angiogenesis and type 3 is involved in the formation of lymphatic vessels. At the same time, while type 1 receptor has a higher affinity to the VEGF, its tyrosine kinase activity is much lower than that of type 2 receptor, which is regarded as one of the regulatory mechanisms preventing an excessive VEGF activity. Accordingly, it is through type 2 receptor that the VEGF effects are normally realized [10, 11].
Upon VEGF interaction with a specific type 2 receptor, autophosphorylation of its intracellular tyrosine sites (Y951, 1054, 1059, 1175, 1214) of kinase and carboxyterminal domains takes place [11]. This in turn activates a number of intracellular proteins, such as phospholipases Cγ, Cβ3, SRK, NCK, SHB, SCK adaptor proteins and others, which comprise first units of the signal transduction complex cascades modifying the morphofunctional state of the target (mostly endothelial) cells. In particular, phospholipase Cγ hydrolyzes the PIP2 membrane phospholipid resulting in the formation of diacylated glycerol and inositol-1,4,5-triphosphate increasing the intracellular content of calcium, which together activate protein kinase C, which in turn triggers sequential activation of the RAS-ERK signal path leading to induction of mitosis. As a result, the proliferative activity of endothelial cells becomes higher [10].
Phospholipase Cβ3 is involved in polymerization of actin and in the formation of stress fibrils enabling migration and motor performance of cells in general [12]. VEGF blocks apoptosis via activation of the “phosphoinositide-3-kinase—protein kinase B” (PI3K/AKT) signal path, inhibiting caspases 3, 7, and 9 and thereby increasing cell survival rate.
In addition, with the help of calcium ions, the PI3K/AKT axis modulates the activity of endothelial NO-synthase, which is accompanied by the increased NO production and increased vascular permeability being a necessary part of angiogenesis (FIG. 3) [13, 14]. Therefore, VEGF, via the specific type 2 receptor, induces activation, migration and differentiation of endotheliocytes and their precursor cells, increases cell survival rate, which in combination with the modulation of intercellular interactions and the increase of vascular permeability provide a prerequisite for the formation of capillary-like constructs followed by their remodeling into “mature” vessels [10-16].
Considering the role of VEGF as a key angiogenic factor, variations of the genes encoding VEGF are used for developing gene constructs suitable for treating patients with cardiovascular diseases of ischemic genesis [17, 18]. In addition, a non-specific angiogenic effect of such preparations for gene therapy may have a positive influence in case of other pathological conditions requiring activation of a reparative process, e.g., injury of peripheral nerves [19], diabetic foot syndrome [20], amyotrophic lateral sclerosis [21], injuries of skeleton bones [22, 23] and others.
As to bone tissue, both in case of primary and secondary osteohistogenesis, precisely the vessels growing into loose fibrous connective or cartilaginous tissue create required conditions for differentiation of the resident cells in the osteoblastic direction as well as for migration of cambial reserves (perivascularly and with the blood flow). Apart from the angiogenesis-mediated influence, VEGF also has a direct stimulating effect on osteoblastic programmed differentiation cells which not only produce VEGF [24] but also express its types 1 and 2 receptors both during embryogenesis [25] and postnatal development [26]. It has been shown that VEGF enables a considerable increase (up to 70%) in proliferation of osseous tissue cambial cells and also activates migration of osteogenic cells according to a VEGF concentration gradient [27-29].
In the past few years, a principally different mechanism referred to as “intracrinic” has been identified in addition to the canonical receptor mechanism of the VEGF action. These findings have been confirmed by the showing that progenitor cells committed in the osteoblastic direction (expressing Osx) synthesize VEGF not only “for exportation” but also to enable its own differentiation in the osteoblastic direction [30].
VEGF has a wide spectrum of action on the endothelial and mesenchymal cell lines. Although a major biological effect of VEGF is associated with induction of the formation of blood and lymphatic vessels, other mechanisms of direct action on the cells of various programmed differentiations via receptors and intracrinic mechanisms may be also characteristic for VEGF. However, more convenient and effective ways of expressing VEGF for treating subjects with diseases, disorders or conditions in need of VEGF are desired.
Conventional Preparations for Gene Therapy
Pharmaceutical preparations for gene therapy comprising nucleic acid constructs and genes expressing useful products, such as vectors containing one or more polynucleotides encoding a therapeutic protein (e.g., VEGF), are becoming of ever greater importance in modern medicine. Until now, five such preparations have been already registered and introduced in clinical practice and hundreds are undergoing experimental studies and clinical trials. A total number of gene therapy clinical trials conducted since 1989 exceeds 1,900 [1]. One of the five registered preparations for gene therapy, Neovasculgen, has been developed and introduced in clinical practice in Russia (Reg. Certificate No. LP-000671 of Sep. 28, 2011) and Ukraine (Reg. Certificate No. 899/13300200000 of Jan. 25, 2013) by the present Applicants. Other gene therapy medicaments are currently under development.
However, existing compositions for gene therapy suffer from a variety of technical problems. While preparations using plasmid vectors to carry a gene encoding a therapeutic protein are considered among the safest types of gene constructs, the efficacy of a gene therapy composition is a function of transformation efficiency, stability of a vector once transformed into a cell, transcription of RNA, stability of transcribed mRNA encoding the therapeutic protein, and activity and stability of the expressed therapeutic protein.
The Neovasculgen gene therapy preparation employs a plasmid DNA vector in combination with a polynucleotide encoding VEGF, a therapeutic protein. However, in order for such gene therapy to be effective a sufficient number of transgenes must enter target cells and be expressed. However, a recognized problem with gene therapy employing plasmid vectors is the low transfection efficiency. Only 1 to 2% of the total number of transgenes reach and/or are expressed by target cells [2]. The low transfection efficiency impacts the amount of therapeutic protein that can be expressed after conventional gene therapy using plasmid vectors because the total number of transfected host cells directly affects the total amount of therapeutic protein produced. On the other hand, for safety and to avoid side-effects it is desirable to minimize the amount of transgenic DNA administered to a subject because administration of too high an amount of transgenic nucleic acid can result in toxicity, inflammatory responses, and in problems with transgene control and targeting.
Consequently, and particularly with respect to gene therapies using plasmids having low transfection efficiencies, methods for increasing the amount of a therapeutic protein encoded by transgenes while decreasing the administered dose of transgenic polynucleotides are of great interest.
There are two principle approaches to increasing the amount of a therapeutic protein expressed by a transgene: (i) modifying the transgenic nucleic acid to improve its uptake by host cells (increasing transfection efficiency) and (ii) increasing the amount of therapeutic protein expressed by host cells that have been transformed with a transgenic nucleic acid. To date there have been significant problems with both approaches.
To increase transfection efficiency, a number of genetic, physical and chemical methods have been proposed. Many or most of these conventional laboratory methods of increasing transfection efficiency, are not generally suitable for gene therapy in the clinic. For example, in the therapeutic context, use of non-plasmid vectors, such as viral vectors, is associated with a significant risk of genetic disruption to a host cell when viral vectors unpredictably integrate into the host cell genome. Physical methods such as direct injection or bombardment of a host cell with particles containing a transgene require special equipment and are not feasible, convenient or safe in a clinical setting. Chemical methods, such as use of liposomes, dendrimers or other chemical agents to facilitate uptake of a transgene by a host cell often result in only transient expression of the transgene and require comingling of the transgene with additional chemical components that create an additional risk when administered to a subject.
The second approached mentioned above seeks to prolong the lifetime of mRNA or other transcription product of a transgene that has entered a host cell, resulting in an increase in the number of times or cycles that mRNA encoding a therapeutic polypeptide is translated, and an overall increase in the amount of the therapeutic product produced by a transfected cell. How mRNA can be stabilized inside the cell to transcribe more of a protein of interest is not completely understood and the cell may require a coordinated system of mRNA degradation and stabilization for normal functioning. According to a number of researchers, some pathological conditions of inflammatory or oncologic genesis may be associated with the post-transcriptional deregulation leading either to insufficient or excessive production of growth factors, oncogenes and other biologically active substances [4]. Thus, a safe and effective method for modulating the mRNA lifetime should attempt to minimize intervention as a way of reducing a risk of disrupting the normal cell functioning.
The prior research of mRNA lifetime has identified some factors including 3′-UTR sequences and regulatory molecules that modulate mRNA lifetime. Regulatory molecules include RNA-binding proteins and regulatory RNAs, such as micro RNAs and long non-coding RNAs. Such regulatory molecules can act via binding to a 3′untranslated region (“3′-UTR”) of protein-coding mRNA. Regulatory molecules once bound to a 3′UTR can destabilize or stabilize mRNA encoding a protein of interest. A number of regulator molecules, such as adenine-uridine-rich-(“AU-rich”) elements, RNA-binding protein 1 (AUF1), tristetraprolin (TTP), KH-type splicing regulatory protein (KSRP) can induce mRNA degradation by binding to 3′UTR specific sites. In contrast, regulatory elements such as polyadenylate-binding protein-interacting protein 2 (PAIP2) stabilize mRNA [3, 4]. The binding of regulator factors to mRNA is only one aspect of the system of regulating mRNA lifetime.
Methods for modifying a 3′UTR nucleotide sequence or a destabilizing element in a 3′UTR were known. These include use of site-specific mutagenesis to modify these sequences. However, to date, it has been unknown whether particular mutations to the 3′-UTR sequence of VEGF would permit one to prolong the lifetime of mRNA encoding VEGF without reducing translation of VEGF or otherwise diminishing expression of VEGF [3]. This unpredictability is due, in part, mRNA isoforms differing in their 3′UTR nucleotide sequences which can vary within a wide range due to alternative splicing, differential polyadenylation and other intracellular mechanisms. Moreover, a composition of a 3′UTR destabilizing (or stabilizing) element also qualitatively and quantitatively depends on its associated nucleic acid coding region. Furthermore, modeling of the effects of particular modifications on mRNA lifetime has been difficult or impossible because the 3′UTR plays other roles in cellular physiology and metabolism. Thus, modification of a 3′UTR can unpredictably affect other cellular processes necessary for stable and prolonged transcription of mRNA and stable, prolonged and active expression of the protein encoded by the mRNA. Consequently, whether a particular modification would increase or decrease mRNA lifetime of mRNA encoding VEGF has previously been unknown.
Despite all difficulties and insufficiently studied issues of the determination or “programming” the mRNA lifetime via changing a 3′UTR sequence, attempts are still being made to empirically select such changes in respective regions of specific gene construct variants that would enhance mRNA stability without negatively affecting its functionality. In particular, a method for increasing transgene production by substituting the AU-rich element sequence presented by AUUUA with other variants and combinations thereof limited to AUGUA, AUAUA, GUGUG, AGGGA, GAGAG, has been known [5]. However, the above sequence of the destabilizing element in 3′UTR is not specific to for all genes (described by most of researchers for G-CSF) and, on the other hand, it does not exhaust a list of the destabilizing elements and, therefore, the mRNA lifetime extension may be diminished. A special 3′UTR sequence of the erythropoietin gene has also been developed that provides prolonged production of the transgene being a part of plasmid DNA. The sequence is strictly specific and has a length of 100 nucleotides [6].
Methods for elimination of specific 3′UTR sequences that are responsible for binding to various microRNAs and that induce mRNA degradation have been also described [7]. However, most of the proposed changes in the 3′UTR sequence relate to lengthy deletions or substitutions, which are inevitably associated with a risk of negative effects on the mRNA metabolism. In addition, proposed solutions are highly dependent on the gene coding region so that some of such solutions are inapplicable while other solutions are insufficiently efficient for a VEGF gene and for extension of the lifetime of its transcription product.
In view of the importance of treating diseases, disorders or conditions that would benefit from transgenic administration of VEGF, the inventors have diligently investigated and now found ways to modify the 3′UTR of VEGF in a way that minimizes a risk of disrupting mRNA regulation in a host cell, while providing for prolonged expression of active VEGF. Moreover, as shown herein, these modifications are effective in a variety of cells including HEK293 human cell line, multipotent mesenchymal stromal cells, and human fibroblasts. To provide this therapy, the inventors diligently studied ways to avoid the problems associated with existing gene therapies, such as therapies that depend on plasmid vectors that have low transfection efficiencies.
The inventors provide herewith a safe and effective way of introducing DNA encoding VEGF into host cells that prolongs VEGF-transgene expression for use in medicinal preparations and gene-activated medical products intended for treating patients not only with cardiovascular diseases but also with other pathology, wherein local increase in the VEGF level within the affected area would enhance the reparative process.